Nowadays, a recombinant engineering technique has been used for producing a variety of physiologically active substances as a recombinant protein in many kinds of host cells including an animal cell and a procaryotic cell such as E. coli. The animal cell is selected as a host cell in case that the protein produced after translation must be subjected to a modification procedure such as glycosilation or a construction of a hyperstructure so that the protein exhibits a physiological activity. However, in order to obtain a large amount of the physiologically active substance which is secreted in a culture supernatant of animal cell, several problems must be overcome. One is of the culture medium for culturing an animal cell capable of continuously producing a desired protein. Another is of the production efficiency of the desired protein in a culture medium in the production thereof by an animal cell.
In case of an animal cell culture, a culture medium must contain serum such as fetal calf serum (FCS). However, the serum including FCS is extremely expensive and besides it may possibly be contaminated with mycoplasma, virus and the like, and hence, it must be assayed for proving no contamination before use. In addition, it has not been easy to purify the desired substance from a culture supernatant since serum contains a variety of heterologous pertinacious substances. In this point of view, a serum-free culture medium has been developed including those supplemented with a protein whose property is well known such as insulin, transferrin, serum albumin and the like [D. Bames, G. H. Sato, Cell, 22 p649 (1980)].
However, it is more preferable not to use any protein. For such a purpose, it is also proposed to replace these proteins by other chemical compounds.
As an alternative of transferrin, an iron chelating agent such as ethylenediamine tetraacetate-iron complex or iron gluconate has been developed [Japanese Patent First Publication (Kokai) NO. 141584/1988; The 8th symposium of the industrial basal technology for the next generation, preliminary report in biotechnology]. As an alternative of serum albumin, a clathrate compound of .alpha.-cyclodextrin with an unsaturated fatty acid or a fat-soluble vitamin is known [Japanese Patent Second Publication (KoKoKu) No. 018465/1981]. It is also reported that an animal cell can be cultured without serum by adding a microemulsion of PLURONIC F-68 and a fat-soluble factor to a culture medium (PCT WO90/03429). However, the above-mentioned culture media without or with low level of protein are directed to merely cell growth and sometimes are not suitably applicable to the production of a recombinant protein. Therefore, there is a need for developing a culture medium which is capable of enhancing the production efficiency of recombinant protein.
As for the production efficiency of culture, in case of human coagulation Factor VIII, it is known that the Factor VIII is present in a starting plasma at a concentration of about 200 ng/ml but when a coagulation Factor VIII-producing cell prepared by the genetic engineering technique is cultured in a usual manner, only about 20 ng/ml of the product can be obtained from the culture supernatant [Pavirani, A. et al., Biotechnology 5, p. 389-392 (1987)]. Such a low level of production in case of the genetic engineering technique is assumed to be mostly due to a large molecular weight of the desired protein (about 300 KDa) and thereby difficulty of secretion of said protein from the host cell to the culture supernatant [Kaufman, R. J. et al., Mol. Cell Biol. 9, p1233-1242 (1989)].
As mentioned above, in case of culture of the Factor VIII-producing cell prepared by the genetic engineering technique, the production efficiency of the desired coagulation Factor VIII in the culture supernatant is quite low by the conventional culturing procedure. In this respect, for enhancing the production efficiency of the recombinant coagulation Factor VIII, an increase of cell density, an increase of stability of the produced protein and an enhancement of expression efficiency must be attained.
The present inventors have been investigated a culture medium which is effective for a large scale production of the recombinant coagulation Factor VIII protein by an animal cell. The present inventors have already found that the production efficiency of the recombinant protein with very low stability such as the coagulation Factor VIII can be improved by adding 1% serum albumin to the culture medium (Japanese Patent Application No. 157570/1988). This is assumed to be due to an effect of the serum albumin to stabilize the produced protein. However, the addition of pertinacious substance such as serum albumin makes it quite difficult to purify the desired production. Besides, since serum albumin is precious and expensive, it is substantially hard to use serum albumin as a culture component in large scale production.
In order to overcome the above-mentioned problem, a number of methods have recently been reported to enhance the production efficiency of the desired protein by adding a kind of additives to a cell culture system. Such known methods include, for example, a method in which the production efficiency of the desired protein is enhanced by adding an alkane acid (the most preferable is butyric acid) or a salt thereof to a culture system of protein-producing cell [Japanese Patent First Publication (Kohyo) No. 503273/1988] and a method in which butyric acid or a salt thereof is added to a beads culture system of protein-producing cell to enhance the production efficiency (PCT WO89/06686). There have also been reported a method for culturing an interferon-producing cell in which a cell capable of intrinsically producing interferon is cultured in a culture medium containing a straight chain alkane acid having 2 to 5 carbon atoms, preferably butyric acid, or a salt thereof prior to induction of interferon production (USP 4,216,203) and a method for enhancing an interferon production which comprises adding glucocorticoid, butyric acid or dimethylsulfoxide to a culture medium [Japanese Patent First Publication (Kokai) No. 74093/1982] and the like.
The above-mentioned conventional methods are directed to enhancement of the production efficiency by adding a straight chain alkane acid, most preferably butyric acid, to a culture system for production of useful protein and in fact the production efficiency of protein is enhanced by these methods through addition of such additives. However, in case of a certain combination of a desired protein and a producing cell, the production efficiency is quite low even when the alkane acid is added to a culture medium and a single addition of butyric acid is even more insufficient. A typical example of such insufficiency is the production of coagulation Factor VIII by utilizing the genetic engineering technique.
Actually, it is reported that, when a recombinant animal cell capable of producing the coagulation Factor VIII prepared by using the genetic engineering technique is cultured to produce a desired protein, the addition of a butyrate (in this case, sodium butyrate) to the culture medium increases an amount of said coagulation Factor VIII produced in the culture supernatant by about 1.3 to 2.5 times as compared to the culture medium with no addition of the butyrate [Dorner, A. J. et al., J. Biol. Chem. 254, p20602-20607 (1989)]. However, the enhancement of production efficiency is still insufficient even by the addition of the butyrate in case of a production system having originally low production efficiency such as the production system of the coagulation Factor VIII, and still more enhancement of the production efficiency is required for the production of coagulation Factor VIII on industrial scale.